The objective of this research project is to determine the functional significance of the glial pathway in the glutamine cycle. The glutamine cycle has been proposed as a mechanism for the efficient reutilization of the transmitters glutamate and GABA by comparmentalization of their metabolism in glia and neurons. The steps that constitute the glial component of the cycle are accumulation of extracellular glutamate and TABA by astrocytes, conversion of these transmitters to glutamine and its subsequent release. For the cycle to operate, glia must produce glutamine at a rate sufficient to resupply the neurons with substrate. Although glutamine synthetase, located exclusively in astrocytes, has been studied in brain and glial homogenates, virtually nothing is known of the function and regulation of this pathway in intact cells. The specific aims of this project are: 1) to quantitate the conversion of glutamate and GABA to glutamine in intact astrocytes, 2) to determine the biochemical and neuronal factors which may regulate this pathway, and 3) to test it as a potential site of therapeutic drug action. Since this pathway is considered crucial for maintaing homeostatic levels of glutamate and GABA, replenishing neuronal metabolic requirements, and detoxifying ammonia, it may be involved in abnormal glutamate or GABA transmission, excitotoxicity, hypoglycemic coma, neurodegenerative disorders, and hepatic encephalopathy. Since certain anticonvulsants interfere with GABA metabolism and several drugs bind with high affinity to astrocytes, their specific effects on the glial pathway may reveal their mechanism of action. To achieve these aims, rat cortical astrocytes in primary cell culture will be incubated with 14C(U)-labelled glutamate, GABA or alpha-KG. Using HPLC with fluorescence detection and radioisotopic procedures, the intracellular and extracellular levels of endogenous and labelled substrates and products will be measured. Studies in this lab indicate that the astrocyte cultures provide the only available intact cell system capable of efficiently carrying out the entire glial pathway from extracellular glutamate to extracellular glutamine. There is preliminary evidence that astrocytes, provided GABA and alpha-KG, produce detectable levels of succinic semialdehyde, the transamination product of GABA. Since these cells do not synthesize GABA, this finding indicates that at least two steps in the glial pathway for GABA, uptake and transamination, operate in astrocyte cultures.